Protein templated synthesis of metal nanoparticles

PROTEIN TEMPLATED SYNTHESIS OF METAL NANOPARTICLES
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Protein Templated Synthesis of Metal Nanoparticles
Arguably, biomolecules assisted production of inorganic nanoparticles, which can be classified into two main categories. For instance, multi-domain protein cages/ templates and self-assembly of biomolecules consisting of small peptides as well as denatured proteins. Therefore, protein cages synthesis of different nanomaterials is relatively comprehended as the templates of biological macromolecules. In other words, their interaction with inorganic ions ultimately determined the size as well as crystallinity of the nanomaterial.
Importantly, the formation of Nanoparticles using protein is not demonstrated by many researchers. However, the preparation of highly crystalline 3-5nm gold nanoparticle relies on the systematic thermal denaturation of protein as well as the protein combination of Escherichia coli in the absence of reducing agent.
Therefore, the use of Uv-vis spectroscopy enabled the protein to determine the size and crystallinity of the nanoparticles. In simple terms, the kinetic of nanoparticle formation modelled the autocatalytic progression. In other words, protein capped which prepared Au nanoparticle typically acts as the catalyst in the stimulation minimization of 4-nitrophenol in the presence of NaBH4. Therefore, these methods demonstrated above is significance in protein assisted synthesis of organic nanostructures particles.
Below is the structure of protein-templated synthesis of metal based nanomaterial.

Figure 1.1 protein template synthesis of metal nanomaterial

Figure 1.1.2 shows the one pot synthesis of two sized clusters for ratio metric sensing of Hg2+
Importantly, spectroscopy is one of the best and efficient methods showing the lysozyme conformational variations. These are characterised in the peak band of 1700-1600 cm-1 and 1480-1575 respectively.

Figure 1.1.3 spectroscopy of lysosomes protein
Therefore, the findings encapsulate how proteins are used in the synthesis of nanoparticles. The reason why protein is used in the production of the nanoparticle is because of their 3 – D structures and unique self – assembling properties (1, 2). Wang et al. (3) cited that these inorganic nanoparticles are capable of nucleation due to the 3 – D structures of proteins and specific amino acid patterns of various peptides that allow these nanoparticles to grow.
Concerning research done by Yang et al. (2) encapsulate that the properties of these nanoparticles, such as surface chemistry, various shapes and sizes are modifiable in using different structures of proteins or arrangement of amino acids in the peptides. Accordingly, the proteins are also used in Metal Nanoparticles synthesis since they provide various chemical groups and binding sites that enable further alteration of these nanoparticles. In simple terms, chemical groups influence nanoparticle alteration based on the fact that during the hydrothermal synthesis, the growth of metal oxides nanoparticles proceeds by the dehydration reaction between surface hydroxyl groups and metal hydroxides. Therefore, these modification allows hybridized metal oxide nanoparticles with biomolecules, polymers as well as surfaces to have strong chemical bonds.
Similarly, protein also utilizes their full functional capabilities, such as providing structural support as well as acting like enzymes or carriers; hence, capable of nanoparticle synthesis (4). The primary role of proteins is to bind metal ions using the various metallic binding sites, for instance, N – terminal amines and Cys residues (5). In other words, binding of protein is based on the interaction between an electron-donating group present on the protein surface and metal ion presenting one or more accessible coordination sites. During synthesis, proteins slowly show their large and overlapping grid Nano cages. For instance, gold, silver and copper nanoclusters (6). Metal sulphides are also formed directly using the same technique, for instance, nickel, copper, and cobalt sulphides. Other metal oxides could also be formed, such as manganese oxides and gadolinium oxides (5).
According to the study of Leng et al. (7), illustrates that the ability to alter functional groups of amino acid residues within peptides gives the most significant potential for forming nanoclusters. The phenolic group present in tyrosine can be changed to provide phenoxide ion by introducing sodium hydroxide, which reduces gold ions to gold nanoclusters. Nanomaterials are widely used in detecting molecular biomarkers of numerous diseases such as neurodegenerative infections, cancers, and others, which are often released into the body fluids. Great potential lies in developing bimetallic nanoparticles for use as catalysts during amplification (8). Potential are individual particles based on the duration of the resistive pulse signals. They are important in nanoparticles because they indicate how stable a particle is within the suspension. One of the examples of the potential is zeta potential.
Importantly, the metal nanoparticles are extensively used in biomedical research and applications. This is due to their smaller size to volume ratios and perfect thermal stability (7). Drug delivery through nanoparticles is efficient in the treatment of malignant tumours such as brain tumours. Gold nanoparticles extracted from carboxyl and alcohol groups permit conjugation with other antibodies, for instance, in the case of Escherichia Coli. Gold nanoparticles are also used in enzyme immobilization to provide inert within the system. Gold particles have lower levels of toxicity and are easy to detect as compared to platinum during medical and biological applications (8).
Protein cavities are hollow spherical, which are formed from several peptides. Protein cages are primarily homogenous in size and their structures. This property enables their interior cavities to function as ideal templates in encapsulating and synthesizing nanoparticles (9). The importance of using protein cage (cavity), as a significant factor in the synthesis of nanoparticles, is to provide a single candidate technique to achieve a uniform particle size. Another advantage of the protein cavity is molecular recognition and metal coordination.

Below diagram shows the hollow structure of the protein cavities

Figure 1.2 hollow cavities of the protein
Advantages of Protein Templates
Size – Renal Clearable
These nanoparticles have optimal clearance from the body, which minimizes the toxic levels of Ag, NPs present in the body by lowering the duration of exposure to such agents (10, 11). Some of the properties affecting renal clearance include; type of particle material, size, and shape of the particle, surface charge, and chemistry. All the factors stated above affects renal clearance in different ways. Concerning the size and shape of the significantly decreased the renal clearance of the particles thus they likely to shift other routes from the kidney to the liver. However, charge related adsorptions due to protein of pure anionic or cation increase hydrodynamic diameter to >15 nm. Hence, it affects the renal clearance. Renal excretion is a desirable path for eliminating nanoparticles from the body by minimal breakdown to lower their side effects. However, the renal approval of nanoparticles is a diverse means that constitutes glomerular filtration, tubular secretion, and lastly, elimination through urinal secretion (11).

Figure 1.3: Renal clearance of various nanoparticles of distinct shapes and charges (11).
In figure 1.3, the circulating particles enter the glomerular capillary wall through the afferent artery of the kidney. This wall has three regions: podocyte regions that extend from the glomerular epithelial cells, negatively charged glomerular basement membrane (GBM), and the fenestrated endothelium. The glomerular filtrate is permitted to flow through the fenestrated endothelium, across the GBM and finally through filtration slits as a result of spaces produced by podocyte extensions (11). The main drawback due to size is the filtration slit with a physiologic pore size of 4.5 nm – 5.0 nm. Nanoparticles less than 6 nm (usually red) are significantly small to be perfectly filtrated irrespective of their inherent charges.
Glomerular filtration of particles of 6 nm – 8 nm in size depends on the charge interactions between particles of intermediate sizes and negative charges of GBM. Thus, positively charged particles are quickly filtered as compared to their equivalent negatively charged particles. As a result of size limitations, nanoparticles larger than 8 nm are not filtered through glomerular filtration. Importantly, the large-sized particle is filtered out through the active process within the cells. Once glomerular filtration is complete, the filtered nanoparticles enter the proximal convoluted tubule (B). In this tubule, nanoparticles may be resorbed from the luminal spaces. Due to brush order (negative charge) of the proximal convoluted tubule epithelial cells, positive charges are readily resorbed from the liminal spaces (10).
Catalytic and Optical Properties
The optical properties of nanoparticles depend on their sizes, and this enables them to provide distinct colours when absorbed in the visible region. Normally, the colour we perceived the object is caused by the photo-degradation. This means that the light of objects is centred on chemical bonds as well as the intensity of the light, which is absorbed by the particular wavelength. For instance, Gold (Au) is wine red, Platinum (Pt) is yellowish-grey, Silver (Ag) is black, and Palladium (Pd) is exceptionally dark, each of 20 – nm. Figure 2 shows gold nanoparticles produced by various sizes; they show different colours as well as properties when their sizes and shape are varied.
However, as it is stipulated in the diagram gold shows different colours depending on the size of the material used to analyse it. The properties of colloidal gold nanoparticles depend on the size and shape of the apparatus used. This is because the transverse and longitudinal absorption peak and anisotropy of the shape affect their self-assembly. Therefore, the suspension of gold of different sizes determines the colour.

Figure 1.3.4: Color variation for Gold (Au) nanoparticles on size and shape (10).
Zhou et al. (12) stated that such optical properties are significant in photocatalytic applications by photo – chemists. Photochemical processes are based on beer – lambert laws and basic principles of light. The distinct colours of various nanoparticles enable them to be used in numerous photo-related applications.
Yang et al. (13) described that catalytic properties of 0 – D and 1 – D nanoparticles have resulted in increased efficiency of these particles, which have saved on production costs. Due to larger surface areas, protein templated nanoparticles have higher catalytic activities with reduced stability over time. Their stability reduces due to the increased ability to undergo agglomeration forming nanoparticles. Agglomeration is more prone in 0 – D structures as compared to 1 – D structures; the latter structures are thus less prone to instability as a result of various uses. Based on the overall shape and structure of nanoparticle material can make them be grouped as 0D, 1D, 2D, 3D. The shape of these materials can influence the glomeration due to variation in the size and shapes.
Facile Synthesis
The ability of these protein templates to undergo facile synthesis is a significant advantage in that it is an environmentally friendly and relatively cheap technique (3). Facile synthesis enables metals that are water-soluble to be successfully extracted and transferred into an organic solvent that floats on water. For instance, LaF3 ∶ Ln3+nanocrystals Water-soluble lanthanide-doped LaF3 nanocrystals. This technique reduces production costs, increases catalytic efficiencies, and results in higher stabilities of produced nanoparticles. Facile wet chemical synthesis (colloidal synthesis) is easy to use with such proteins and has numerous environmentally friendly paths. The whole process involves constant growth, coalescing of crystals, growth of shells/cores, and finally, galvanic replacement. In the comparison of facial and traditional “wet chemical” technique is a long-established approach and provides various several advantages compared to traditional. One of the advantages is that facial wet does not result in aggregates and agglomerates as shown by the traditional pyrolysis
. Its environmentally friendly feature allows it to be scaled for large scale processing in meeting commercial and industrial needs (13).

Figure 1.4.1 facile synthesis of the protein
Examples of Protein Synthesis of Metal Nanoparticles and Their Uses
Gold
Large scale production of gold nanoparticles is realized through reduction technique— green reducing agents of proteins which are usually activated by hydrogen peroxide and superoxide anion (7). Collagen proteins usually convert chloroauric acid to aqueous gold anions, which are further reduced by sodium borohydride and sodium citrate to form gold nanoparticles of distinct sizes ranging from 2 nm – 40 nm. The produced GNPs are monodispersed and usually spherical (8). GNPs from distinct proteins are usually in the visible {1 0 0} facets, for instance, MSN–Proteins–GNPs Nano-Bioconjugate, have different ultraviolet absorption spectra and numerous colours, as shown in figure 3.

Figure 1.5: Large scale reduction of synthesizing monodispersed and spherical shaped gold nanoparticles using proteins (8).
In figure 3 (a), the whole reduction process is visualized, and the reducing power which is initiated by hydrogen peroxide and superoxide anion is further examined.
Uses
Gold nanoparticles are widely used as sensors in detecting biological molecules due to their plasmonic properties (9). Over the years, thin gold layers are used in producing surface Plasmon resonance detectors, which are employed in sensing binding between any biological molecules, for instance, the presence of antibody-antigen binding. Plasmon resonance detectors occurs when the polarised light strike an electrically conducting surface at the interface between the two media. This action generates electron charge density waves known as Plasmon, reducing the intensity of inflected light of reflected light at specific angle. These detectors allow medical professionals to obtain real-time measurements in solutions to resembling physiological situations.
GNPs are also widely used in surface-enhanced Raman spectroscopy based on the chemical nature of the biological molecules, which can be examined through Raman scattering of laser light (10). Signals generated by gold nanoparticles are enhanced numerous orders of magnitude as a result of localized surface Plasmon resonance effects produced on the surface of the nanoparticle.
Copper
According to Ramyadevi et al. (15), biological extracts used for copper synthesis are obtained mainly from microbial extracts. Cu NPs are also produced from higher angiosperm plants.
The mechanism of Cu NP formation is due to the bioreduction of metal nanoparticles through combining biomolecules present in plant extracts (proteins and amino acids) and the corresponding phytochemical (15). Phytochemicals (flavones and aldehydes) are significant in the instant reduction of these nanoparticles. Example of the phytochemical used are polyphenols and flavonoids. Microorganisms’ form Cu NPs by grabbing target ions present on the surface or inside microbial cells, reduces these ions to form nanoparticles in the presence of enzymatic actions.
Enzymatic action

Figure 2.5 enzymatic action in Cu NPs formation
Copper nanoparticles are widely used as antibacterial and antimicrobial agents. Copper has a higher affinity for oxygen and its oxides are very stable under thermodynamic conditions. The development of this oxide layer on the surface decreases its antimicrobial properties. These nanoparticles kill bacteria by hitting numerous targets, this disrupts the outer membranes of the bacteria including exchange of ions and other substances. Once the outer membranes are disrupted, pathways for cell death are created to destroy the bacteria (14, 15).

Figure 1.6 biological extract of copper synthesis
Silver
Silver nanoparticle (AgNPs) can be synthesized with different protein as a template through the introduction of the traditional light. This is because it silver possesses distinct localized surface of Plasmon resonance with absorption spectra that can be synthesised by different protein during the irradiation by the light. Therefore, protein templated synthesis of silver nanoparticles occurs through microorganisms. Numerous microorganisms are used, including the most unaffected prokaryotic cells to eukaryotic fungi and plants. By mediating fungal proteins from Aspergillus fumigatus, silver nanoparticles are made. It is realized by 60% per cent volumes of cell-free filtrate and 2.0 mM of silver nitrate solution. A pH of 10.0 is usually maintained for 90 minutes to attain maximum efficiencies. However, to achieve this process, different organism named above acts catalyst in catalysing the process.
Silver nanoparticles have antibacterial properties that have been utilized in numerous applications to control the growth of bacteria, for instance, dental surgery and related applications, treatment of burns and wounds. When silver nanoparticles are introduced in wounds and burns, there is a greater reduction in scar appearance as opposed to silver sulfadiazine, since they suppress both local and systemic tissue inflammations. These nanoparticles promote the rate of wound closure by proliferating and adjusting the keratinocytes. Wound contraction is facilitated by differentiating fibroblasts into myoblasts. Silver ions and related compounds are highly toxic for any microorganisms to withstand (16).
Due to their broader antimicrobial spectrum, they are widely used in sterilization of consumer and medical products, for example, in the textile industry, manufacture of food storage bags, refrigeration surfaces, and also products for personal use. Since all bacteria have enzymes in the form of “chemical lung” for a breakdown of oxygen, antibacterial properties of silver nanoparticles are seen when they cripple these enzymes by inhibiting them from taking up oxygen and later they suffocate within 5 minutes without affecting adjacent tissues. They are also used in optical analyses for surface-enhanced Raman scattering as well as metal improved fluorescence. They have more significant extinction coefficients, better field enhancements, and optimum extinction bands (17). Other applications include colourimetric sensing for the design of silver-based fluorescent sensors with higher sensitivity. These colourimetric detectors are usually coupled with localized surface Plasmon resonances to effectively and quickly identify ions such as mercury. They are also employed as catalysts in redox reactions involving fast dye reduction as well as Methyl Orange degradation processes. The diagram below summarised all the process of the silver formation

Figure 1.90 AgNPs formation
Platinum
Protein templated synthesis of platinum was demonstrated by Behravan et al. (18), in which a leaf extract of Diospyros kaki was utilized as the reducing agent. Platinum ions are reduced to form platinum nanoparticles after the leaf extract was exposed to an optimum temperature of 95 degrees Celsius.
Platinum nanoparticles are characterized as antimicrobial, antioxidant, and anticancer properties, and using Fourier Transform Infrared (FTIR) to allow the location of significant biomolecules in reduced platinum ions as well as the stable formed platinum nanoparticles (19). Platinum nanoparticles are widely used in the synthesis of electrocatalysts as well as catalytic converters, manufacture of magnetic Nanopowders, membranes of polymers, cancer therapies, and development of coatings.
Platinum nanoparticles utilized in toxicity and therapeutic evaluations, usually have cancerous cells and malignant tumours as their biological target sites. Once these nanoparticles enter cells, they destroy DNA and produce antioxidant responses. The DNA integrity is compromised resulted to a further reduction in cellular glutathione (20).

Figure 1.7 synthesis of platinum nanocages by the aid of liposomes containing photocalyst molecules
Palladium
The preparation of various plant extracts achieves protein templated synthesis in palladium. Examples of plant extracts used include leaves or whole plants. This is because biosynthesis of palladium nanoparticle from P glutinosa plant extract has been done at ninety after stirring the mixture PdCl2 + extract for two hours. Therefore, a change in colour shows the formation of the palladium nanoparticles which was confirmed by the presence of the Uv light.
Mechanism reaction

Figure 2.5 mechanism reaction of the palladium
Once these extracts are prepared, a bioreduction and further fabrication to form a metallic solution occur (21). These nanoparticles are analysed further using ultraviolet spectroscopy and finally characterized by either scanning electron microscopy or transmission electron microscopy.
These particles show a characteristic brown colour due to a positive two valence state. A biosynthesis conducted from distinct plant extracts, for instance, P. glutinosa for 90 degrees at 120 minutes, produces a significant change in colour ranging from light yellow to dark brown. Colour change signifies that the nanoparticles have been successfully formed through the reduction process (21, 22). The results of protein templated synthesis of palladium nanoparticles are characterized by a TEM micrograph, which revealed that these nanoparticles were enclosed within an organic layer obtained from the extract. In this way, it acts as both capping and reducing agents. Mostly nanoparticles of 20 – 25 nm in size are produced. The IR spectrum gives the presence of flavonoids as well as polyphenols.
Palladium is extensively used in oxidative addition as well as reductive elimination of hydrogen. They are also employed in catalysis of organic cross coupling reactions in which palladium salts having neither phosphines nor any added ligands are used for reaction, activation of electrodeless metal deposition, oxidation of primary alcohols when it is uniformly spread on conductive materials in alkaline solutions, and hydrogenation of unsaturated olefins. Due to its high affinity for hydrogen, palladium is used in the development of hydrogen fuel cells, hydrogen sensors, hydrogen storage, amongst other roles (22).
 
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